Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ.

Phosphorylation of elongation factor 2 (EF-2) by specific Ca2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3]. / Affinnaia modifikatsiia 40S subchastits iz platsenty cheloveka proizvodnymi pAUG i pAUGU3.

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.

Interaction of wheat germ translation initiation factor 2 with GDP and GTP.

The wheat germ translation initiation factor 2 (WGeIF-2) was isolated in a homogeneous state by an efficient procedure and characterized. Its molecular mass, as determined by a gel-filtration method is approximately 150,000 Da. According to SDS-PAGE WGeIF-2 consists of four subunits with M(r) 37,000 (alpha), 40,000 (beta), 42,000 (gamma) and 52,000 (delta). The beta- and gamma-subunits (but not the alpha-subunit) of WGeIF-2 can be readily phosphorylated by the double-stranded RNA activated kinase isolated from rabbit reticulocytes. Dissociation constants for WGeIF-2 complexes with GDP and GTP were measured. In our evaluation the WGeIF-2 affinity for GDP (KdGDP = 1.5 x 10(-7) M) was only 10 times higher than for GTP (KdGTP = 1.5 x 10(-6) M), while for rabbit reticulocyte eIF-2 (RReIF-2) the difference has been estimated as as much as two orders of magnitude in accordance with the literature. Close values of dissociation constants for WGeIF-2 complexes with guanine nucleotides suggest that at a sufficiently high [GTP]/[GDP] ratio the nucleotide exchange in wheat cells may take place without the participation of specific factor (eIF-2B) which catalyzes the nucleotide exchange on eIF-2 from mammalian cells.

Eukaryotic protein synthesis initiation factor 2. A target for inactivation by proanthocyanidin.

Polyproanthocyanidin (PPA), a phenolic polymer isolated from the plant Alhagi kirgisorum S. was found to interact strongly with eukaryotic initiation factor 2 (eIF-2), thereby inhibiting reactions involving this protein. When added to a rabbit reticulocyte lysate system, PPA blocks in vitro translation and it appears to selectively bind and precipitate a relatively small number of proteins including eIF-2 and regulin. The phosphorylation of purified regulin and eIF-2 by casein kinase II (CK II) and the heme-sensitive eIF-2 alpha kinase, respectively, was also inhibited by the polyphenolic compound. The natural fluorescence of PPA was utilized to compare its interaction with eIF-2 and regulin to that with other natural and synthetic polypeptides.

An inhibitor of protein synthesis initiation from Alhagi kirgisorum S.

Polyproanthocyanidin--a plant phenolic compound from Alhagi kirgisorum S. effectively inhibited protein synthesis in rabbit reticulocyte and wheat germ cell-free systems. Poly-proanthocyanidin inhibited translation only at the level of initiation and not at the elongation level and aminoacylation of tRNA. The inhibitory effect of the phenolic compound is due to the blockage of the ternary complex formation of eIF-2 with GTP and initiator Met-tRNA.

The excess GTP hydrolyzed during mistranslation is expended at the stage of EF-Tu-promoted binding of non-cognate aminoacyl-tRNA.

The system of translation of Sepharose-bound poly(U) in which all ribosomes are active in peptide elongation was used to determine the stoichiometry of GTP hydrolysis at the stage of EF-Tu-promoted aminoacyl-tRNA binding. The ratio of GTP hydrolyzed at this stage per peptide bond was assayed during codon-specific elongation (polyphenylalanine synthesis) and misreading (polyleucine synthesis). It was demonstrated directly that the excess GTP hydrolyzed during misreading [(1984) FEBS Letters 178, 283-287] is expended at the stage of Ef-Tu-promoted binding of non-cognate aminoacyl-tRNA.

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system.

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.

Stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system. Determination of GTP/peptide bond ratios during codon-specific elongation and misreading.

The stoichiometry of GTP hydrolysis during peptide elongation in the processes of codon-specific translation and misreading of polyuridylic acid was determined in a cell-free system in which all ribosomes were active in peptide synthesis. Ribosomes carrying oligophenylalanine presynthesized on poly(U) covalently bound to Sepharose were used. In the codon-specific translation of poly(Phe) on poly(U)-Sepharose at optimal Mg2+ concentration (6 mM MgCl2), the ratio of GTP cleaved to Phe polymerized was found to be about 2 (+/- 0.1). Under the same conditions but during misreading (elongation of polyleucine on poly(U)-Sepharose) the GTP/Leu ratio increased 10 times (from 16 to 25 in different experiments).

[Stoichiometry of GTP breakdown during peptide synthesis on the ribosome. Stoichiometry of GTP hydrolysis during elongation of polyphenylalanine on polyuridylic acid]. / Izuchenie stekhiometrii raspada GTP pri sinteze peptida na ribosome. Stekhiometriia gidroliza GTP v protsesse élongatsii polifenilalanina na poliuridilovoi kislote.

The stoichiometry of GTP hydrolysis during poly(Phe) elongation by Phe on poly(U) covalently bound to Sepharose was determined. The concentrations of both EF-T and EF-G were saturating. The GTP/Phe stoichiometry was calculated without the usual correction for the uncoupled ribosomal EF-T and EF-G dependent GTP hydrolysis. At the Mg2+ optimum (6 mM) for the poly(Phe) elongation on poly(U) . Sepharose the stoichiometry ratio of GTP/Phe was 1.9/2.1. This indicates that two (or less) GTP molecules coupled with poly(Phe) elongation by Phe on poly(U) . Sepharose are hydrolyzed.

[Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. GTP hydrolysis uncoupled with ribosomal peptide synthesis and dependent on preparation of elongation factor T]. / Izuchenie stekhiometrii raspada GTP pri sinteze peptida na ribosome. Gidroliz GTP, ne sopriazhennyi s sintezom peptida i zavisimi ot preparata faktora elongatsii T.

It is shown that preparations of EF-T are practically free from GTPase activity, if they were formed from EF-Tu and EF-Ts carefully purified from GTPase beforehand. Some Ef-T-dependent GTPase can arise after addition of tRNA or aminoacyl-tRNA preparations to a EF-T+ribosomes mixture. Poly(U) stimulates, as a rule, EF-T-dependent GTPase in the EF-T/ribosomes+tRNA mixture. Uncoupled EF-T-dependent GTPase of the EF-T+ribosomes+tRNA+poly(U) mixture is by 1--2 orders of magnitude less than uncoupled EF-G-dependent GTPase under the same conditions. A conclusion is made that uncoupled EF-T-dependent GTPase is an inevitable obstacle when studying the GTP expenditure in the process of ribosomal protein synthesis, if a traditional cell-free protein-synthesizing system is used for this purpose.

[Stoichiometry of GTP hydrolysis during peptide synthesis on the ribosome. I. Factor-independent GTPase and ATPase of ribosomal preparations]. / Izuchenie stekhiometrii raspada GTP pri sinteze peptida na ribosome. Faktornezavisimaia GTPaza i ATPaza preparatov ribosom.

It has been found that preparations of Escherichia coli (MRE-600) ribosomes can display GTPase and ATPase activities independent of elongation factors EF-Tu and EF-G. The GTPase and ATPase are localized on ribosomal 50S subparticles, whereas 30S subparticles are free of the activities and do not stimulate them upon association with the 50S subparticles to form complete ribosomes. The GTPase and ATPase can be removed from the ribosomes and their 50S subparticles by treatment with 1 M NH4Cl or 50% ethanol in the cold. Ribosomal preparations freed from the factor-independent GTPase and ATPase retain their basic functional features. The data obtained do not permit to solve finally whether the factor-independent GTPase and ATPase revealed are components of ribosomes or represent a contamination rather firmly bound to the ribosomes. However, in any case this finding can contribute to an uncoupled hydrolysis of GTP and should be considered when studying the stoichiometry of triphosphate expenditure in the process of ribosomal protein synthesis.